In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
Mass spectrometry (MS)-based proteomics has become a method of choice to study proteins in a global manner (1-3). Mass spectrometry is not inherently quantitative but methods have been developed to address this limitation to a certain extent. Most of them are based on stable isotopes and introduce a mass shifted version of the peptides of interest, which are then quantified by their ‘heavy’ to ‘light’ ratio. Stable isotope labeling is either accomplished by chemical addition of labeled reagents, enzymatic isotope labeling or metabolic labeling (4-6). Generally, these approaches are used to obtain relative quantitative information on proteome expression levels in a light and a heavy labeled sample. For example, stable isotope labeling by amino acids in cell culture SILAC (7, 8) is performed by metabolic incorporation of differently labeled, such as light or heavy labeled amino acids into the proteome. Labeled proteomes can also be used as internal standards for determining protein levels of a cell or tissue proteome of interest, such as in the spike-in SILAC approach (9).
Absolute quantification is technically more challenging than relative quantification and could so far only be performed accurately for a single or a small number of proteins at a time (10). Typical applications of absolute quantifications are the determination of cellular copy numbers of proteins (important for systems biology) or the concentration of biomarkers in body fluids (important for medical applications). Furthermore, any precise method of absolute quantification, when performed in more than one sample, also yields the relative amounts of the protein between these samples.
Several methods for absolute quantification have emerged over the last years including AQUA (11), QConCAT (12, 13), PSAQ (14), absolute SILAC (15) and FlexiQuant (16). They all quantify the endogenous protein of interest by the heavy to light ratios to a defined amount of the labeled counterpart spiked into the sample and are primarily distinguished from each other by either spiking in heavy labeled peptides or heavy labeled full length proteins. The AQUA strategy uses proteotypic peptides (17) which are chemically synthesized with heavy isotopes and spiked in after sample preparation. AQUA peptides are commercially available but expensive, especially when many peptides or proteins need to be quantified (see, for example, Kettenbach et al., Nat. Protoc. 2011, 6:175-86). Moreover, the AQUA strategy suffers from quantification uncertainties that are introduced due to spiking in of the peptide standard after sample preparation and enzymatic proteolysis, which is a late stage in the workflow. Furthermore, any losses of the peptides—for example during storage—would directly influence quantification results. The QconCAT approach is based on artificial proteins that are a concatamers of proteotypic peptides. This artificial protein is recombinantly expressed in Escherichia coli and spiked into the sample before proteolysis. QconCAT allows production of labeled peptides, but does not correct any bias arising from protein fractionation effects or digestion efficiency. The PSAQ, absolute SILAC and FlexiQuant approaches try to address these limitations by metabolically labeling full length proteins by heavy versions of the amino acids arginine and lysine. PSAQ and FlexiQuant synthesize full-length proteins in vitro in wheat germ extracts or in bacterial cell extract, respectively, whereas absolute SILAC was described with recombinant protein expression in E. coli. The protein standard is added at an early stage, such as directly to cell lysate. Consequently, sample fractionation can be performed in parallel and the SILAC protein is digested together with the proteome under investigation. However, these advantages come at the cost of having to produce full length proteins, which limits throughput and generally restricts these methods to soluble proteins.
Accordingly, there is an unmet need for improved or alternative means and methods of mass spectrometry-based absolute quantitation of peptides and polypeptides.